1,3-Propanediol has utility in a number of applications, including as a starting material for producing polyesters, polyethers, and polyurethanes. Methods for producing 1,3-propanediol include both traditional chemical routes and biological routes. Biological methods for producing 1,3-propanediol have been recently described (Zeng et al., Adv. Biochem. Eng. Biotechnol., 74:239-259 (2002)). Biologically producing 1,3-propanediol requires glycerol as a substrate for a two-step sequential reaction. First, a dehydratase (typically a coenzyme B12-dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde (3-HP). Then, 3-HP is reduced to 1,3-propanediol by an NADH- (or NADPH) dependent oxidoreductase (See Equations 1 and 2).Glycerol→3-HP+H2O  (Equation 1)3-HP+NADH+H+→1,3-Propanediol+NAD+  (Equation 2)The 1,3-propanediol is not metabolized further and, as a result, accumulates in high concentration in the media.
Typically, glycerol is used as the starting material for biologically producing 1,3-propanediol. However, glucose and other carbohydrates also are suitable substrates for 1,3-propanediol production. Specifically, Laffend et al. (WO 96/35796; U.S. Pat. No. 5,686,276) disclose a method for producing 1,3-propanediol from a carbon substrate other than glycerol or dihydroxyacetone (especially, e.g., from glucose), using a single microorganism comprising a dehydratase activity. Emptage et al. (WO 01/012833) describe a significant increase in titer (grams product per liter) obtained by virtue of a non-specific catalytic activity (distinguished from the 1,3-propanediol oxidoreductase encoded by dhaT) to convert 3-HP to 1,3-propanediol. Payne et al. (U.S. No. 60/374,931) disclose specific vectors and plasmids useful for biologically producing 1,3-propanediol. Cervin et al. (U.S. No. 60/416,192) disclose improved E. coli strains for high yield production of 1,3-propanediol. WO 96/35796, WO 01/012833, U.S. No. 60/374,931, and U.S. No. 60/416,192 are incorporated by reference in the instant specification as though set forth in their entirety herein.
The enzymes responsible for converting glycerol to 3-HP are largely coenzyme B12-dependent enzymes, known as coenzyme B12-dependent glycerol dehydratases (E.C. 4.2.1.30) and coenzyme B12-dependent diol dehydratases (E.C. 4.2.1.28). These distinct, but related, coenzyme B12-dependent enzymes are well studied in terms of their molecular and biochemical properties. Genes for coenzyme B12-dependent dehydratases have been identified, for example, in Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, Klebsiella oxytoca, and Lactobacillus collinoides (Toraya, T., In Metalloenzymes Involving Amino Acid-Residue and Related Radicals; Sigel, H. and Sigel, A., Eds.; Metal Ions in Biological Systems; Marcel Dekker: New York, 1994; Vol. 30, pp 217-254; Daniel et al., FEMS Microbiology Reviews 22:553-566 (1999); and Sauvageot et al., FEMS Microbiology Letters 209: 69-74 (2002)).
Although there is wide variation in the gene nomenclature used in the literature, in each case the coenzyme B12-dependent dehydratase is composed of three subunits: the large or “α” subunit, the medium or “β” subunit, and the small or “γ” subunit. These subunits assemble in an α2β2γ2 structure to form the apoenzyme. Coenzyme B12 (the active cofactor species) binds to the apoenzyme to form the catalytically active holoenzyme. Coenzyme B12 is required for catalytic activity as it is involved in the radical mechanism by which catalysis occurs.
Biochemically, both coenzyme B12-dependent glycerol and coenzyme B12-dependent diol dehydratases are known to be subject to mechanism-based suicide inactivation by glycerol and other substrates (Daniel et al., supra; Seifert, et al., Eur. J. Biochem. 268:2369-2378 (2001)). In addition, inactivation occurs via interaction of the holoenzyme with high concentrations of 1,3-propanediol. Inactivation involves cleavage of the cobalt-carbon (Co—C) bond of the coenzyme B12 cofactor, leading to the formation of 5′-deoxyadenosine and an inactive cobalamin species. The inactive cobalamin species remains tightly bound to the dehydratase; dissociation does not occur without the intervention of coenzyme B12-dependent dehydratase reactivation factors (“dehydratase reactivation factors”). This inactivation can significantly decrease the reaction kinetics associated with 3-HP formation and, thus, indirectly decrease 1,3-propanediol production.
The effects of coenzyme B12-dependant dehydratase inactivation can be partially overcome. For example, inactivation can be overcome by relying on those proteins responsible for reactivating the dehydratase activity. Dehydratase reactivation factors have been described in: WO 98/21341 (U.S. Pat. No. 6,013,494); Daniel et al. (supra); Toraya and Mori (J. Biol. Chem. 274:3372 (1999)); and Tobimatsu et al. (J. Bacteria 181:4110 (1999)). Reactivation occurs in a multi-step process. Initially, interaction of inactivated coenzyme B12-dependent dehydratase with dehydratase reactivation factors, in an ATP-dependent process, results in the release of the tightly bound inactive cobalamin species to produce apoenzyzme. Subsequently, the dehydratase apoenzyme may bind coenzyme B12 to re-form the catalytically active holoenzyme and the inactive cobalamin species may be regenerated (by enzymatic action, in a separate ATP-dependent process) to coenzyme B12. Depending solely on dehydratase reactivation factors to restore dehydratase activity is inherently limited, however, since both the dehydratase reactivation and the coenzyme B12 regeneration processes require ATP. These ATP-dependent processes represent a significant energetic burden to the process of converting glycerol to 3-HP, particularly if a subsequent reaction of 3-HP to 1,3-propanediol is present and the 1,3-propanediol concentration is high.
Alternatively, it is possible to either increase the amount of coenzyme B12 added to a medium during 1,3-propanediol production or to supplement the culture media with vitamin B12 (which is converted to coenzyme B12 in vivo) to supply additional coenzyme B12 to microorganisms. However, in both cases, the cost of these additions may significantly interfere with process economics.
Croux et al. (WO 01/04324 A1) have addressed the problems associated with coenzyme B12-dependent dehydratases by developing a process for producing 1,3-propanediol using a recombinant microorganism that expresses a coenzyme B12-independent dehydratase. However, the usefulness of this solution may be limited by the ability of B12-independent dehydratases to function under certain preferred process conditions (e.g. under aerobic conditions (Hartmanis and Stadman, Arch. Biochem. Biophys. 245: 144-52 (1986)).
In principle, it should be possible to isolate coenzyme B12-dependent dehydratases with reduced inactivation kinetics from naturally occurring microbial strains. Reduced inactivation kinetics would increase the turnover ratio (mol product/mol holoenzyme) of coenzyme B12-dependent dehydratase in a microbial host, and thus, reduce the dehydratase and coenzyme B12 demand. This approach would also reduce the energy needed to maintain that level of dehydratase and coenzyme B12. However, in practice, the diversity of coenzyme B12-dependent dehydratases has been found to be limited with respect to inactivation kinetics.
The problem to be solved, therefore, is that currently available coenzyme B12-dependent dehydratase enzymes are unable to provide the reaction kinetics needed for industrial applications for the production of industrial compounds.